PROJECT SUMMARY The overall objective of this project is to develop a nanofiber-hydrogel composite that is capable of organizing differentiated cells from the transplanted human pluripotent stem cell (hiPSC)-derived neural/progenitor stem cells (NSCs) at the lesion site following brain injury, and enhancing the integration of the regenerated neurons with host tissue. A growing body of evidences has suggested exogenous stem cell transplantation is one promising strategy to promote injured brain tissue regeneration. However, the ongoing inflammation at the lesion site and the lack of supportive tissue structure and vasculature within the traumatic lesion cavity present a hostile environment that result in low cell survival and poor control over differentiation and engraftment of the transplanted stem cells. We have recently shown that an optimized hyaluronic acid (HA) hydrogel conjugated with laminin-derived peptide as a cell-delivery matrix with the ability to bind and enrich the endogenous angiogenic factors, such as vascular endothelial growth factor, generated a robust neovascular network within the hydrogel at the traumatic lesion cavity. Human fetal tissue-derived NSCs delivered in this HA hydrogel have shown enhanced survival following implantation at the lesion site, and the majority of survived NSCs has differentiated into neuronal progenitors and populated the entire lesion cavity. However, these neuronal progenitors exhibited disorganized structure at the lesion site with limited integration with host cortex tissue. Since an ordered and layered structure is important to the function of the brain cortex, control over cell organization and integration following NSC transplantation and differentiation at the lesion site is critical to the success of stem cell-based therapy for brain injury. Here we hypothesize that the differentiated cells from transplanted hiPSC-derived NSCs can be guided to form aligned and organized structure by a 3D nanofiber- hydrogel composite in brain lesion site, which in turn facilitates the integration of the regenerated neurons with host tissue. To test this hypothesis, we will first develop a nanofiber-hydrogel composite with aligned nanofibers and demonstrate its ability to organize NSCs during differentiation and maturation. The nanofiber- hydrogel composite will be tailored with adhesive cues, optimized pore size and modulus to support human iPSC-derived NSCs in directing their migration and promoting their differentiation and maturation (Aim 1). We will then transplant the optimized composite scaffold to demonstrate the advantages of our approach in promoting structural repair of the brain tissue and ensuing functional improvement in neurological outcomes (Aim 2). This composite with defined compositions is highly desirable for clinical translation.